Proportion Of Leukemic Cells Research Articles

Maintenance therapy following CD19 CAR-T treatment for relapsed B-cell acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation

目的探讨异基因造血干细胞移植后复发急性B淋巴细胞白血病（B-ALL）CD19 CAR-T细胞治疗缓解后的维持治疗，观察单独输注供者造血干细胞和供者T淋巴细胞对CD19 CAR-T细胞扩增的影响。方法1例难治B-ALL患者，CD19 CAR-T（鼠源，克隆号：FMC63）细胞治疗后桥接同胞全相合异基因造血干细胞移植，后再次复发。接受CD19 CAR-T（可变区人源化的FMC63）细胞治疗再次缓解，缓解后先后接受单独输注供者造血干细胞、供者T淋巴细胞的维持治疗，期间监测患者细胞因子水平、CD19 CAR-T细胞比例、CD19 CAR的DNA表达变化，观察骨髓白血病细胞比例及供者嵌合情况。另外，通过制备小鼠CD19 CAR-T（FMC63）细胞，并将CAR-T细胞注射至同窝小鼠体内，14 d后，再次注射同窝小鼠的B淋巴细胞，在此过程中观察CAR-T细胞、B淋巴细胞比例以及CD19 CAR的DNA表达，以此来模拟临床治疗过程。结果①患者输注人源化CD19 CAR-T细胞后第14天，疗效评估达完全缓解（CR），供者嵌合率为99.76%，细胞因子释放综合征1级。②人源化CD19 CAR-T细胞治疗缓解后，单独输注供者造血干细胞维持治疗，输注后第24天出现移植物抗宿主病（GVHD），患者GVHD期间伴有外周血CD19 CAR-T细胞比例及CD19 CAR DNA水平的升高，并随GVHD缓解下降。过程中患者维持CR且供者嵌合率为99.69%。③随后单独输注供者T淋巴细胞维持治疗，回输后第12天患者出现GVHD，而患者外周血CD19 CAR-T细胞比例及CD19 CAR的DNA水平并未出现再次升高。过程中患者维持CR且供者嵌合率为99.87%。④C57小鼠体内试验证实，CAR-T细胞输注后小鼠体内CAR-T细胞扩增及DNA表达上调，同时CD19+ B淋巴细胞耗竭。之后输注CD19+ B淋巴细胞，CD19 CAR-T细胞扩增和CD19 CAR的DNA表达再度上调。结论供者造血干细胞、供者T淋巴细胞的单独输注，可作为异基因造血干细胞移植后复发B-ALL接受CD19 CAR-T治疗缓解后的维持治疗手段，且输注供者造血干细胞诱发GVHD同时出现CD19 CAR-T细胞和CD19 CAR的DNA表达上调，有可能进一步清除残留灶。

Role of Vascular Niche of Bone Marrow in the Development of MLL-AF9 Induced Acute Myclaid Leukemia

To investigate the role of bone marrow vascular niche in the development of MLL-AF9 acute myeloid leukemia (AML). Transplantation experiments were performed to establish non-radiated MLL-AF9 AML model; the half-bone immunofluorescence staining and tile-scan imaging of two-photon confocal microscopy were used to obtain the data of 3 main bone marrow niche cells; flow cytometry analysis was performed to characterize leukemia cells in different anatomical sites. In the early stage of MLL-AF9 AML, the proportion of leukemia cells in the metaphysis of the femur was significantly higher than that in the diaphysis. The detection of apoptosis and proliferation rate of leukemia cells showed that the percentage of leukemia cells in metaphysis significantly decreased, and the proliferation (S/G2/M phase) was also significantly more active. These different features of leukemia cells may relate with different bone marrow microenvironment. The image data of 3 major components of bone marrow niche (endothelial cells, endosteum, megakaryocytes) showed different distribution of blood vessels in metaphysis and diaphysis. Furtherly comparing the spatial distance between leukemia cells and endothelial cells, endosteum, megakaryocytes indicated that leukemia cells are closer to the blood vessels, suggesting the important role of blood vessels in the development of leukemia. Glucose uptake assays and intracellular ROS detection showed that the supportive role of blood vessels for leukemia cells did not related with nutrient metabolism pathway. The vascular niche plays an important role in the development of leukemia, and does not relate with the transport of nutrients and the elimination of metabolic waste, instead, which may relate with perivascular cytokines or other vascular functions.

Abstract CT060: A clinical study on CD33-directed chimeric antigen receptormodified NKcells in patient with refractory or relapsed acute myeloid leukemia

Abstract In patients with acute myeloid leukemia, an about 20-40% of the patients show no response to current therapy, and about 50-70% of the patients relapse after complete response (CR). These patients represent the majority of the refractory or relapsed acute myeloid leukemia (R/R AML). The 1-year survival rate of those patients is limited to <30% and it further drops to approximately 10% for 5-year survival. More effective treatment for this disease is urgently needed. As a new type of cell-based immune therapy, chimeric antigen receptor modified NK (CAR-NK) cell treatment is now promising in treating hematological malignancies. CD33 is found to be expressed at higher levels on both AML cells and leukemia stem cells (LSCs) compared with normal hematopoietic stem cells (HPSCs). Therefore, use of CD33-directed chimeric antigen receptor modified NK cells (CD33-CAR-NK) may lead to the development of a novel method for the treatment of R/R AML patients. In this study, three patients with R/R AML expressing high level of CD33 (40-70%) were enrolled in a clinical study; two were M1, and one was M2a. All three patients had received a series of standard treatments with no therapeutic effect before 3 x 109 allogeneic CD33-CAR-NK cells were infused. Approximately 10% of the infused cells were confirmed to be transduced with the CAR. The protocol was to infuse 1 x 109 CD33-CAR-NK cells and repeated every other day for three times. After CD33-CAR-NK cell infusion, the main side effects include repeated low-grade fever and significant rises of serum cytokines, e.g. IFN-γ, TNF-α, IL-2, IL-6 and IL-10. However, before discharge, the level of the cytokines reduced to normal. In addition, while we detected K+, Ca2+, creatinine and glutamic-pyruvic transaminase after CAR-NK infusion, all of these were not significantly changed compared with those before the CAR-NK infusion. These results suggested that there was no significant tumor necrosis after treatment. After CD33-CAR-NK cell infusions, the proportion of leukemic cells in the patient's bone marrow decreased significantly. For example, in one of the two AML-M1 patients, a 53 year-old male, the proportion of leukemic cells in bone marrow reduced from 40% to 26% after CD33-CAR-NK cell infusion. In addition, the proportion of the CD33+ cells in bone marrow reduced from 48.82% to 32.56%, and the CD33+CD34+ cells in bone marrow reduced from 40.59% to 27.26% respectively. This initial clinical work indicates that CD33-CAR-NK therapy may offer a new therapeutic option for R/R AML, which aims to reduce the tumor burden, eliminate LSCs, and provide an opportunity to combine its use with other therapies for more effective treatment of the R/R AML patients. Key words: Refractory or relapsed acute myeloid leukemia (R/R AML), CD33, Chimeric antigen receptor (CAR), NK cells, Immunotherapy. Citation Format: Leiming Xia, Xiang Sun, Liu Liu, Yi Wang, Tan Li, Bin Li, Gregory Wolf, Qiao Li, Lin Yang, Yangyi Bao. A clinical study on CD33-directed chimeric antigen receptormodified NKcells in patient with refractory or relapsed acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT060.

Chemosensitizing AML cells by targeting bone marrow endothelial cells.

Refractory disease is the greatest challenge in treating patients with acute myeloid leukemia (AML). Blood vessels may serve as sanctuary sites for AML. When AML cells were co-cultured with bone marrow endothelial cells (BMECs), a greater proportion of leukemia cells were in G0/G1. This led us to a strategy of targeting BMECs with tubulin-binding combretastatins, causing BMECs to lose their flat phenotype, degrade their cytoskeleton, cease growth, and impair migration despite unchanged BMEC viability and metabolism. Combretastatins also caused downregulation of BMEC adhesion molecules known to tether AML cells, including vascular cell adhesion molecule (VCAM)-1 and vascular endothelial (VE)-cadherin. When AML-BMEC co-cultures were treated with combretastatins, a significantly greater proportion of AML cells dislodged from BMECs and entered the G2/M cell cycle, suggesting enhanced susceptibility to cell cycle agents. Indeed, the combination of combretastatins and cytotoxic chemotherapy enhanced additive AML cell death. In vivo mice xenograft studies confirmed this finding by revealing complete AML regression after treatment with combretastatins and cytotoxic chemotherapy. Beyond highlighting the pathologic role of BMECs in the leukemia microenvironment as a protective reservoir of disease, these results support a new strategy for using vascular-targeting combretastatins in combination with cytotoxic chemotherapy to treat AML.

Data and implication based comparison of two chronic myeloid leukemia models

Chronic myeloid leukemia, a disorder of hematopoietic stem cells, is currently treated using targeted molecular therapy with imatinib. We compare two models that describe the treatment of CML, a multi-scale model (Model 1) and a simple cell competition model (Model 2). Both models describe the competition of leukemic and normal cells, however Model 1 also describes the dynamics of BCR-ABL, the oncogene targeted by imatinib, at the sub-cellular level. Using clinical data, we analyze the differences in estimated parameters between the models and the capacity for each model to predict drug resistance. We found that while both models fit the data well, Model 1 is more biologically relevant. The estimated parameter ranges for Model 2 are unrealistic, whereas the parameter ranges for Model 1 are close to values found in literature. We also found that Model 1 predicts long-term drug resistance from patient data, which is exhibited by both an increase in the proportion of leukemic cells as well as an increase in BCR-ABL/ABL Model 2, however, is not able to predict resistance and accurately model the clinical data. These results suggest that including sub-cellular mechanisms in a mathematical model of CML can increase the accuracy of parameter estimation and may help to predict long-term drug resistance.

Detection of occult cerebrospinal fluid involvement during maintenance therapy identifies a group of children with acute lymphoblastic leukemia at high risk for relapse

We aimed at assessing the clinical significance of the levels of acute lymphoblastic leukemia (ALL) cells in samples of cerebrospinal fluid (CSF) during therapy. We studied 990 CSF samples from 108 patients, at the time of diagnosis (108) and at each time of intrathecal therapy (882). The proportions of leukemic cells in CSF samples were assessed by flow cytometry (FCM). Patients with central nervous system (CNS) involvement at diagnosis (FCM+) showed predominantly a T-ALL, and higher percentages of known negative prognostic factors: high risk group, higher white blood cell counts, normal karyotype, and the BCR-ABL fusion gene. No differences in relapse free survival (RFS) and overall survival (OS) were observed between FCM+ versus FCM- at diagnosis. Patients with CNS involvement during therapy showed significantly older age, and higher frequencies of T-cell leukemia. We found a significantly higher RFS in patients with FCM+ during therapy. The detection of subclinical CNS disease by FCM during maintenance was associated with significantly lower 3-years RFS and 3-years OS. A sensitive methodology like FCM can be applied for a close follow-up of the levels of ALL in CFS samples, and may identify a group of patients at high risk for relapse.

178-P

Aim We present the cases of two AML patients HLA typed by rSSO and SBT as homozygous, and subsequently typed as heterozygous by SSP. Methods Potential bone marrow transplant recipients were typed for HLA-A, -B, -C, -DR, -DQ by low resolution rSSO (LabType, One Lambda) and high resolution SBT (SeCore, Life Technologies), using DNA extracted (Qiagen EZ, 20 ng/μl) from EDTA anticoagulated peripheral blood. SSP (Olerup) was used as a secondary method for clarification. Results Patient #1, a 71y.o. female caucasian in AML blast crisis (86% blasts in peripheral blood), was typed by rSSO and SBT. Results at the A locus were discrepant, DR-DQ association was uncommon, and a possible homozygous B ∗ 44:27 warranted confirmation. Further SSP testing to investigate the discrepancy revealed a full heterozygous HLA typing. Buccal cell DNA typed by SBT confirmed heterozygous DQB1 ∗ 05:02 and ∗ 06:04 [ Table 1 ]. Patient #2, a 68 y.o. female diagnosed with AML, was typed by rSSO and SBT. Discrepancies were detected at A and B loci, and the C locus appeared homozygous. SSP method revealed a heterozygous HLA-A, -B, -C typing [ Table 2 ]. Conclusions These two cases reinforce the importance of close scrutiny when reviewing HLA molecular typing results. HLA heterozygous patients may present as homozygous from peripheral blood with large proportions of leukemic cells. We suggest using multiple methods and/or DNA from sources other than peripheral blood to confirm homozygous HLA loci in this patient population.

HLA-G expression in B chronic lymphocytic leukemia: a new prognostic marker?

Chronic lymphocytic leukemia (CLL) is characterized by a malignant clonal population of lymphocytes, which are usually of the B cell lineage. Classical Rai and Binet staging of CLL is being superseded by new prognostic markers. The mutational status of the immunoglobulin variable region heavy-chain genes segregates the disease into more benign and more malignant versions, and has been confirmed as an important prognostic marker in prospective clinical trials. A search for surrogate markers for this assay has led to flow cytometric assays for CD38 and ZAP-70 expression. The human leukocyte antigen G (HLA-G) molecule exhibits limited tissue distribution and a low polymorphism that generate seven HLA-G isoforms. HLA-G exerts multiple immunoregulatory functions. Recent studies indicate an ectopic up-regulation in tumor cells that may favor their escape from anti-tumor immune responses. For this report we studied HLA-G in parallel with CD38 and ZAP-70 in B-cell chronic lymphocytic leukemia (B-CLL) patients. HLA-G expression was studied retrospectively in circulating B-CLL cells from 20 patients by flow cytometry using the anti-HLA-G specific monoclonal antibody MEM/G9. The proportion of leukemic cells expressing HLA-G varied from 1 to 34%. We detected a statistically significant correlation between HLA-G positive (>12%) expression and progression free survival (p=0·045), but no correlation with CD38 and ZAP-70. We also detected a statistically significant difference between Binet stage A; B and C (p=0·046) and a positive correlation between IL-10 and HLA-G (p=0·044). We conclude that positive HLA-G has an effect on progression - free survival, when compared with CD38 and ZAP-70.

FISH analysis for CML monitoring ?

Conventional cytogenetics is considered the gold standard for evaluating CML during interferon (IFN) treatment. Drawbacks to this approach are the small number of metaphases available during IFN therapy and the impossibility of scoring interphase cells. We applied, besides cytogenetics, double-color FISH (dc-FISH) detection of BCR-ABL gene fusion to monitor 20 CML patients on IFN. dc-FISH easily detected 200 cells per specimen, while with cytogenetic examination a mean of 16.1 mitoses per sample were scored. Though the correlation of dc-FISH and cytogenetic data was good (r = 0.77, p < 0.001), the discrepancy between the two methods as regards the proportion of leukemic cells in the marrow was often important dc-FISH detected a relevant proportion of BCR-ABL+ cells in three patients classified as complete cytogenetic responders and showed that, after 9-12 months of IFN treatment, a significant reduction of BCR-ABL+ cells was present in all the 20 patients tested. This might suggest that all CML patients are potentially responsive to IFN. Though more data are required, we think that dc-FISH is more informative than cytogenetic analysis for CML monitoring. Notably because of the simplicity of the procedure, this method could be easily standardized among different laboratories, thus permitting cross-comparison in therapeutic trials.

Liquid culture and cryopreservation of marrow cells of leukaemic patients prior to autologous bone marrow transplantation.

The feasibility of marrow cryopreservation for autologous bone marrow transplantation after 7 d in liquid culture was assessed in 10 leukaemic patients. A median of 0.17 x 10(8) nucleated cells/kg and 0.4 x 10(4) CFU-GM/kg could be collected after the complete procedure, with overall a consistent cell loss. Long-term cultures could be established from these cultured and frozen marrows, showing the persistance of precursors of haematopoietic and stromal cells. In vitro a significant decrease in the proportion of leukaemic cells could be observed in only one out of nine evaluable patients. This patient, with refractory AML, received an autologous transplant and is alive in continuous complete remission after 600 d. One patient with chronic myeloid leukaemia in acute phase underwent an autologous BMT with a marrow collected and cultured while in chronic phase and failed to engraft. These results show the feasibility of cryopreservation of cultured marrow cells for autologous bone marrow transplantation. The procedure is associated with poor cell recovery and must be improved to have a more general clinical application. This technology may have a major application with the emergence of modulators of growth and differentiation of haematopoietic cell lines.

Identification of leukemic cells in the cerebrospinal fluid from children with acute lymphoblastic leukemia: advances and dilemmas.

As more children survive acute lymphoblastic leukemia, new challenges face the pediatric oncologist. One is the need for early recognition of subclinical nonsymptomatic disease, or occult meningeal leukemia. By obtaining spinal fluid specimens, one can frequently monitor the central nervous system (CNS) for the presence of residual or recurrent leukemia. Methods for identifying small numbers of lymphoblasts in the spinal fluid have progressed from subjective descriptions of nucleated cells to assignment of objective methods for labeling the cells in question. These more sensitive methods of detection have challenged the "old" definitions of remission. The unequivocal identification of lymphoblasts in the spinal fluid of a child with less than 5 cells/mm3 of fluid is an example of such a dilemma. Does this finding herald overt CNS relapse or hematologic relapse? Does it warrant further, more aggressive treatment beyond giving standard prophylactic CNS therapy? It can be suggested that the finding of even a small number or proportion of leukemic cells in the spinal fluid in a child being given continuation/maintenance therapy has a high probability of being followed by an adverse event (relapse). If this is true, then the definition of CNS leukemia needs refinement and the methods for unequivocally identifying leukemic cells in a spinal fluid sample depend on techniques of cytologic examination beyond routine cytomorphology. Such laboratory methods are now available, but they require more clinical testing and standardization before their sensitivity and specificity in identifying leukemic cells is established.

DETECTION OF A MULTIDRUG RESISTANT PHENOTYPE IN ACUTE NON-LYMPHOBLASTIC LEUKAEMIA

Most adult patients with acute non-lymphoblastic leukaemia (ANLL) relapse with drug resistance. Overexpression of a plasma membrane protein, P-glycoprotein, correlates with multidrug resistance in human and animal cell lines. We have detected a multidrug resistance phenotype in two patients with drug resistant ANLL by an immunocytochemical assay using a monoclonal antibody to P-glycoprotein. Sequential analysis of peripheral blood samples from both patients showed a progressive increase in both the intensity of staining and the proportion of leukaemic cells that bound antibody as the disease progressed. The assay is simple, and may have prognostic and therapeutic implications.

Low dose cytosine arabinoside (Ara C) in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are acquired bone-marrow disorders, characterized by a decreased ability of the haemopoietic cell to differentiate, resulting in peripheral cytopenias. The majority of patients will die either from acute myeloid leukaemia or from infection and/or haemorrhage. Thirty-eight courses of low dose Ara C were administered in 26 MDS patients. Nineteen courses (50%) were associated with good (12) or partial (7) response. Three complete remissions were observed. The median duration of response overall was 19.5 weeks, 26 weeks for the good and 10 weeks for the partial responders. A high incidence of treatment failure was seen in patients treated after transition to AML. Major complications were observed during 14 courses and mortality was directly related to therapy in five patients. Platelet transfusions were required during 26 courses. Aggravation of peripheral-blood cytopenia during the first weeks and hypocellularity of bone-marrow aspirates at the end of therapy suggest that low-dose Ara C exerts its main activity by suppression of leukaemic growth, rather than by induction of differentiation in malignant cells. Our results in MDS patients demonstrate that low-dose Ara C can be of value in severe cytopenia and can decrease the proportion of leukaemic cells in the bone marrow, but the danger of treatment should not be underestimated.

Effect of schedule on activity and toxicity of 5-azacytidine in acute leukemia: a Southwest Oncology Group Study.

One-hundred-fifty-four patients with acute leukemia and extensive prior chemotherapy were treated with 5-Azacytidine and evaluated according to five different schedules. One-hundred-twenty patients received adequate trials; 34 patients died within 14 days of onset of treatment. Nine patients achieved a complete remission (CR) and two achieved a partial remission. Although two of the treatments have a higher remission rate, the data were not statistically significant. The median time to CR was 48 days (range 21-173). The median duration of CR was 65 days (range 39-369). There was no difference in response rate according to cell type. The median age of responders was 31 years, and 39 years for nonresponders. Proportionately there were more women among responders (5M/6F) and more men (70M/39F) among nonresponders. At onset of therapy the median leukocyte counts were similar between responding (5.4 X 10(3)) and nonresponding (5.7 X 10(3)) patients, but the proportion of leukemic cells was significantly higher among nonresponding patients (46% vs. 7%). Toxicities included nausea, vomiting, diarrhea, skin rash, myalgias, prolonged myelosuppression, hypotension, and central nervous system stupor and/or coma. Lower dose continuous infusion schedules of five-, seven-, and ten-days duration appear effective and were associated with less toxicity.